A series of physical and chemical methods are proposed which are aimed at characterizing the structure and reactivity of the binuclear iron site in an intermediate Fe(II)---Fe(III) oxidation level of the non-heme oxygen-carrying protein, hemerythrin. This intermediate "semi-met" oxidation level could be on the pathway of reversible oxygenation. Thus, its characterization could lead to a clearer understanding of the mechanism of reversible oxygen binding. Experiments in the principal investigator's lab have partially characterized a sulfide derivative of hemerythrin which is at the semi-met oxidation level. Experiments are proposed to thoroughly characterize this derivative with regard to location of sulfide binding and sulfide to iron stoichiometry. Studies of CO and NO derivatives of the protein are proposed which could provide insights into how electrons are transferred to and from as well as within the iron site. Differences in physical and chemical properties have been observed in semi-met hemerythrin prepared from different species and from different directions (i.e. from either fully oxidized or fully reduced hemerythrins.) These differences could arise from conformational constraints due to subunit interactions within the octameric protein. Thus it is proposed to both dissociate and isolate the subunits in order to examine the properties in the absence of even transient subunit interactions. The proposed experiments are designed to provide the foundation for a long term project whose goal is a clearer understanding of reversible oxygen binding by proteins of both vertebrate and invertebrate species.